ABSTRACT Checkpoint blockade immunotherapy relies on releasing tumor-specific T cells from normal inhibitory control, and has significantly impacted treatment for several types of cancer. However, response rates in patients treated with checkpoint blockade still need to be improved. Non-responsiveness to checkpoint blockade could result from many causes, including failure of dendritic cell priming of CD8 T cells. Indeed, checkpoint blockade has recently been shown to be dependent on the cDC1 subset of dendritic cells for its effectiveness. The cDC1 lineage of dendritic cells is the major cross-presenting cell that primes CD8 T cells and has been the subject of our recent molecular and developmental analysis. We were the first to identify that the cDC1 lineage requires the BATF3 transcription factor and is critical in vivo for tumor rejection. Cross-presentation is central to the ability of cDC1 to prime tumor-specific DC8 T cells, but this process has remained poorly understood. We have undertaken a molecular dissection of the mechanisms of cross-presentation in cDC1. Our ra- tionale is that understanding cross-presentation at a basic level could be used to improve treatment of can- cer patients by reducing non-responsiveness to checkpoint blockade. In addition, this could potentially be used to improve dendritic cell vaccination approaches in cancer. This application builds on our discovery of the first gene that is absolutely required for cDC1 cross-presentation in vivo and is critical for anti-tumor re- sponses. We identified Wdfy4 in a CRISPR/Cas9 screen in primary cDC1 as required for cross-presentation and we have already developed the Wdfy4-/- mouse model which is the basis for the current application. Our preliminary data show that Wdfy4-/- mice have a profound defect in cross-presentation, lacking the ability to prime CD8 T cells against viruses and tumors. We propose to use this model to determine the full scope of Wdfy4's function in vivo and to determine the mechanism by which WDFY4 supports cross-presentation in cDC1. Further, we will determine the molecular and cellular mechanism for this function of the WDFY4 protein. First, we will determine the intracellular location of WDFY4 in DCs, by using immunofluorescence microscopy of epitope tagged WDFY4 proteins, by localizing WDFY4 using APEX2 fusion proteins and electron microsco- py, and by localizing endogenous WDFY4 in primary human and mouse cDC1. Second we will identify WDFY4 interacting partners that cooperate in cross-presentation in cDC1 by testing whether Clec9a or Dec205 interact with WDFY4, by identifying WDFY4 interacting proteins by immunoprecipitation and mass-spectrometry analy- sis, and by proximity labeling, and by testing if these interacting proteins are required for cross-presentation.